The inability to functionally repair tissues lost as a consequence of disease or accident remains a significant challenge for regenerative medicine. Tissue engineers have developed various types of replacement scaffolding with the aim of enhancing skeletal muscle regrowth. However, these scaffolds are typically comprised of artificial polymers or decellularized extracellular matrix (ECM) that mimic adult tissues and their success has been hindered by limited host incorporation. What is not commonly taken into consideration by regenerative biologists is that the ECM undergoes dramatic remodeling during development, repair and regeneration, creating an environment that is biomechanically distinct from the homeostatic adult. I hypothesize that by harnessing the basic elements nature uses to assemble the musculoskeletal system in vivo, the functional integration of replacement constructs into the host will be significantly enhanced. The objectives of this proposal are to map the distribution of key ECM during musculoskeletal development and to develop a robust in situ imaging method to elucidate how cell behavior is regulated by ECM composition. I will achieve my objective through the successful completion of the following two specific aims: 1) Create a method to image the behavior of individual cells within the developing mouse forelimb using Cre-lox technology, multiphoton microscopy and murine embryo cultures. Muscle progenitors will be labeled in double mutants that are heterozygous for Pax3-Cre and ROSA-ZsGreen1. By imaging the migration of Pax3+/ZsGreen1+ muscle progenitors from the somites into the developing limb, the parameters for live imaging will be determined and refined. 2) Map the spatial distribution of key ECM during myogenesis and quantify the effect of hyaluronic acid (HA) knockdown on myoblast migration. The organization of key ECM, including HA, will be characterized immunohistochemically in relation to Pax3+ myogenic precursors and differentiating muscle during the early stages of limb development. The influence of HA on muscle progenitor migration will be directly tested by imaging murine embryo cultures in which HA has been pharmacologically (via 4-methylumbelliferone) or genetically (by mating HAS2-floxed mice with cell-specific Cre-expressing mice) knocked down. I expect that the knockdown of HA will affect muscle development by significantly decreasing the distance Pax3+ cells migrate from the somites and resulting in premature differentiation of myogenic precursors. My long-term goal is to use this novel method to assess the effect of perturbations on ECM-cell interactions during musculoskeletal development to identify specific components that regulate muscle assembly. The proposed research is significant because it will 1) create a method to image the response of cells to a wide range of biochemical and molecular interventions in the native 3D environment of the developing forelimb, 2) characterize the role of hyaluronic acid during skeletal muscle development and 3) lead to new tissue engineering approaches for the replacement of damaged muscle.